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Padarti et al. Vessel Plus 2018;2:21 I http://dx.doi.org/10.20517/2574-1209.2018.34 Page 15 of 23

CCM3 stabilizes intracellular bridges
Certain cells such as germ cells have cytoplasmic connections that regulate cell-cell communication and
coordination. Anillin proteins such as ANI-1 and ANI-2 regulate the length of these projections. ANI-1 is
known to decrease bridge length, while its antagonist, ANI-2, increases bridge length . It was found that GCK-1
[167]
that is regulated by CCM3 binds to ANI-1. Therefore, deficiencies in CCM3 and GCK-1 result in a decrease
in intracellular bridge size. This results in multiple histological defects in the gonads such as reduced distal
arm length, rachis diameter, and brood size. Fluorescence imaging studies showed that CCM3 localizes to the
bridges. Co-deletions of CCM3/GCK-1 and ANI-1 resulted in increased bridge number, suggesting a similar
pathway between GCK-1/CCM3 and ANI-1. Non-muscle myosin II (NMMII) is responsible for constriction of
bridges. However, unopposed activation of NMMII causes hyperconstriction and results in destabilization of
bridges . CCM3/GCK1 deletion resulted in increased localization of NMMII to the intracellular bridges and
[168]
ANI-1 binds to NMMII. Therefore, it was postulated that intracellular bridges is regulated by CCM3-GCKI-
ANI-1- NMMII signaling cascade. Yet, co-deletion of these genes did not affect bridge size. Therefore, it is likely
that GCK1/CCM3 affects intracellular bridges through other signaling pathways.

CCM lesions have defective autophagy
CCM3, along with CCM1 and CCM2, are involved in many signaling pathways that result in increased
production of ROS: Sirt1/FoxO1, JNK/c-JUN, β-catenin, and TGF-β pathways [146,169-171] . This oxidative stress
will damage organelles in the cell. However, inadequate autophagy mechanisms hinder cell recovery ability
leading to progression of disease. CCM lesions show defect in autophagy through increased activity of
mTOR. Inhibitors of mTOR were shown to reverse the defect in autophagy suggesting that mTOR is involved
in the process, which provides another set of pharmacotherapeutic agents in CCM.

CCM lesions have differentially expressed miRNA
The composition of micro RNAs (miRNAs) in CCM lesions was analyzed through an mRNA expression screen.
These results were supported by RT-qPCR. Compared to normal controls, it was found that 10 miRNAs were
upregulated and 42 miRNAs was downregulated in CCM lesions. A more stringent analysis showed 5 miRNAs
that were very significantly downregulated. Using bioinformatics, potential binding mRNAs to these 5 miRNAs
were identified. One of the miRNAs had a potential 981 binding partners. Several proteins already implicated
in CCM lesions were found to be targets of these miRNAs including MLLT4, VEGFA, MAPK1, RAC1, RHOA,
FOXO1, ENG, SMURF1, and HEYL [17,83,99,106,126,139] . It was concluded that three miRNAs (let-7b-5p, miR-361-5p,
and miR-370-3p) can potentially be involved in the pathogenesis of CCM .
[172]

CCM3 was frequently implicated in tumorigenesis
CCM3 was initially identified as a tumor-associated apoptotic protein . Several cases of meningiomas
[21]
have been reported in patients with dysfunctional CCM3, suggesting that CCM3 could potentially act as
a tumor suppressor [31,173,174] . One report stated that CCM3 deficient EC cells can continuously proliferate
in cell cultures. In fibroblasts, CCM3 deficient cells can grow several more generations before entering
senescence, comparing to wild-type cells. This suggests that the depletion of CCM3 delays cell senescence.
Gene enrichment analysis showed a decreased production of cytokines in CCM3 deficient EC cells. Cytokine
production was not inducible with TNF-α in these cells. It was found that these cells have a defect in C/EBPβ
activity. C/EBPβ expression was upregulated in CCM3 deficient cells, which delays the progression of cells
into senescence. Therefore, the lack of C/EBPβ activity is the likely driven factor in delaying the cells into
senescence. Gene enrichment analysis showed decreased expression level of lysosome gene set. Senescent
cells have increased autophagy for unutilized organelles and CCM3 deficient cells do not show increased
activity of autophagy. Growth of CCM3 depleted cells in minimum nutrient media showed impairment of
autophagy. Therefore, CCM3 deficiency increases C/EBPβ activity that, in turn, impairs cell senescence,
resulting in declined cellular autophagy . More research is needed to elucidate the underlined relationship
[175]
between CCM3-mediated senescence and meningiomas.